The present invention relates to compounds that mimic peptides. More particularly, the present invention relates to the synthesis of peptides in which xcex1-carbons of the peptide backbone have been replaced by trivalent nitrogen atoms using either solution phase or liquid phase synthetic methodologies.
Peptidomimetics have become immensely important for both organic and medicinal chemists (Spatola et al. Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins; Weinstein, B., Ed.; Marcel Dekker: New York, 1983; pp. 267-357; Sherman et al. J. Am. Chem. Soc. 1990, 112, 433; Hirschmann et al. Angew. Chem. Int. Ed. Engl. 1990, 29, 1278; Gante et al. Angew. Chem. Int. Ed. Engl. 1994, 33, 1699). Synthetic interest in these surrogate peptide structures has been driven by the pharmaceutical industry""s needs for molecules with improved pharmacokinetic properties (Hodgson et al. Bio/Technology 1993, 11, 683). Biophysical studies on these pseudopeptides has allowed elucidation of the functional role of the peptide backbone (Marshall et al. Chemical Recognition in Biological Systems; Creighton et al. The Chemical Society: London, 1982; p 278; Farmer et al. Drug Design; Ariens, E. J., Ed.; Academic Press, New York, 1980, p. 121) and with an ever-increasing level of synthetic sophistication the degree of peptide mimicry within a peptidomimetric can be tailored to chemist""s needs. Indeed, the alteration of peptides to peptidomimetics has included peptide side chain manipulations, amino acid extensions (Freidinger et al. Science 1980, 210, 656; Paruszewski et al. Rocz. Chem. 1973, 47, 735; Stachowiak et al. J. Med. Chem. 1979, 22, 1128), deletions (Rivier et al. Chemia 1972, 26, 303; Sarantakis et al. Clin. Endocrinol. 1976, 5, 2755), substitutions, and most recently backbone modifications (Hagihara et al. J. Am. Chem. Soc. 1992, 114, 6568; Simon et al. Proc. Natl. Acad. Sci. USA 1992, 89, 9367; Smith et al. J. Am. Chem. Soc. 1992, 10 114, 10672; Cho et al. Science 1993, 261, 1303; Liskamp et al. Angew. Chem. Int. Ed. Engl. 1994, 33, 633; Burgess et al. Angew. Chem. Int. Ed. Engl. 1995, 34, 907). It is this latter development that has been exploited for the synthesis of biomimetic polymeric structures. Such progress has been fueled by the suggestion that peptidomimetics may provide novel scaffolds for the generation of macromolecules with new properties of both biological and chemical interest.
The most common manipulation involving the xcex1-carbon atom of peptides is the inversion of stereochemistry to yield D-amino acids (Spatola et al. Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins; Weinstein, B., Ed.; Marcel Dekker: New York, 1983; pp. 267-357). The importance of this substitution in affording compounds with improved biological potencies, altered conformational properties (Mosberg et al. Proc. Natl. Acad. Sci. USA 1983, 80, 5871), and increased resistance to enzymatic degradation has been widely recognized and exploited (Dooley et al. Science 1994, 266, 2019). Replacement of the xcex1-hydrogen of the common amino acids by a methyl group, or by any other substituents (NH2CRRxe2x80x2CO2H) are both further examples of xcex1-alkyl modification. Azapeptides, however, are peptides in which one (or more) of the xcex1-carbon(s) has been replaced by a trivalent nitrogen atom (FIG. 5) (Gante et al. Synthesis 1989, 405). This transformation results in a loss of asymmetry associated with the xcex1-carbon and yields a structure that can be considered intermediate in configuration between D- and L- amino acids (Aubry et al. Biopolymers 1989, 28, 109). Interest in this xcex1-carbon replacement unit stems from its ability to provide resistance to enzymatic cleavage and its capacity to act as a selective inhibitor of cysteine (Magrath et al. J. Med. Chem. 1992, 35, 4279) and serine proteases (Elmore et al. Biochem. J. 1968, 107, 103; Barker et al. Biochem. J. 1974, 139, 555; Gray et al. Tetrahedron 1977, 33, 837; Gupton et al. J. Biol. Chem. 1984, 259, 4279; Powers et al. J. Biol. Chem. 1984, 259, 4288). While the synthesis of azapeptides has been reported (Bentley et al. J. Chem. Soc. (C) 1966, 60; Dutta et al. J. Chem. Soc. Perkin Trans 1 1975, 1712; Furr et al. J. Chem. Soc. Perkin Trans 1 1979, 379; Quibell et al. J. Chem. Soc. Perkin Trans 1 1993, 2843), the synthesis of a xe2x80x9cpure azapeptidexe2x80x9d, or what we will term an xe2x80x9cazatidexe2x80x9d has yet to be accomplished. The earliest attempts to make pure azatides can be dated to Gante and co-workers. (Gante et al. Chem. Ber. 1965, 98, 3340; Gante et al. Proc. Am. Pept. Symp. 13th. 1993, 1994, 299) However, the methodology that was reported does not allow azatide stepwise chain lengthening in a repetitive manner of anything but hydrazine units.
The utility of azatide compounds has been demonstrated in the treatment of various disorders including cancers, viral infections and cataracts. For example, Moretti et al, J. Clin. Endocrino. Metab., 81(11), 3930-3937, 1996, show that azatide compounds are used as LH-releasing hormone agonists to interfere with stimulatory actions of epidermal growth factor in human prostatic cancer cell lines. Jeyarajah et al, Gynecol. Oncol., 63(1), 47-52, 1996, use an azatide gonadotropin-releasing hormone analog for treatment of recurrent endometrial cancers. Brower et al, J. Surg. Res., 52(1), 6-14, 1992, show differential effects of azatide containing LHRH and somatostatin analogs on human breast cancers. Hellberg et al., PCT Int. Appl. WO 9640107 A1 961219, demonstrate the use of (N,Nxe2x80x2-bis(mercaptoacetyl) hydrazine derivatives as anticataract agents. Nakashima et al., EP 672678 A1 950920, show the preparation and use of azapeptide compounds as neurokinin A antagonists. Azatide type compounds have been used as retroviral protease inhibitors, Kempf et al., PCT Int. Appl. WO 9414436 A1 940707, lipoxygenase inhibitors (Atkinson et al Eur. Pat. Appl. EP 146243 A1 850626) and immunosuppressant rapamycin carbamate analogs, Kao et al. U.S. application Ser. No. 5,411,967A 950502.
What is needed is either a solution phase or liquid phase synthetic methodology for synthesizing azatides using monomeric xe2x80x9cxcex1-aza-amino acidsxe2x80x9d which can be coupled in a linear and stepwise chain-lengthening fashion. What are needed are azatides as mimetics for peptides which are are easy to synthesize, more stable and more active than the parent peptides. Moreover, azatide mimetics are needed for stability as compared to various natural peptide products and compounds which possess better bioavailability and exhibit greater activity as compared to known peptides.
The invention is directed to the azatides and a method for the synthesis of azatide mimetics. In particular, an efficient method has been developed for the solution and liquid phase syntheses of biopolymer mimetics consisting of xe2x80x9cxcex1-aza-amino acidsxe2x80x9d linked in a repetitive manner to form an azatide oligomer. A general synthetic procedure is claimed which provides the use and synthesis of a wide variety of Boc-protected aza-amino acid monomers with optimization of solution phase procedures for the coupling of aza-amino acids in a repetitive manner. In addition, the design and synthesis of a linker is employed that supports azatide synthesis using a liquid phase synthetic format. Oligoazatides can now be rapidly assembled on a homogeneous polymeric support. Using the methodology provides a potential source of new peptidomimetic libraries.
One aspect of the invention is directed to a process for synthesizing an oligoazatide. The process employs a support material with a linker unit attached thereto. The preferred support material is a soluble homopolymer support, e.g., polyethylene glycol monomethyl ether (MeO-PEG). Polyethylene glycol monomethyl ether is soluble in aqueous media but precipitates in ether. Precipitation of the support material with ether can be employed for purifying coupled molecules. Alternative soluble supports having this property are caonventional and may be readily substituted for the polyethylene glycol monomethyl ether. Solid phase supports may also be employed but are less preferred because of their poorer yields and/or difficulty in handling.
A linker unit is attached attached to the soluble support. A preferred linker unit is p-hydroxymethylbenzoate. The process also employs a Boc-protected aza-amino acid. Preferred Boc-protected aza-amino acids are represented by the following structure: 
wherein Rx is selected from the group consisting of hydrogen, methyl, isobutyl, isopropyl, C1-C6 alkyl, benzyl, substituted benzyl and the side chain radical of the following amino acids: Ala, Arg, Asn, Asp, Asx, Cys, Gln, Glu, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val. The Boc-protected aza-amino acid is reacted with a carbonyl activation element for producing an activated carbamate of the Boc-protected aza-amino acid. A preferred carbonyl activation element is bis-pentafluorophenyl carbonate. The soluble homopolymer support is then coupled with the activated carbamate for producing a nascent protected chain. The nascent protected chain may then be deprotected using a mild acid for producing a nascent deprotected chain. A preferred mild acid for the deprotection step is trifluoroacetic acid. The nascent deprotected chain is then washed by precipitation of the soluble homopolymer. The nascent deprotected chain may then be extended by repeating the above xe2x80x9cnxe2x80x9d times wherein 1xe2x89xa6n xe2x89xa6100 and wherein the soluble homopolymer support of said Step A is replaced with the nascent deprotected chain for producing an extended deprotected chain. Then, the extended deprotected chain is decoupled from the soluble support by hydrogenation for producing the oligoazatide.
Another aspect of the invention is directed to a process for producing a combinatorial oligoazatide library. The process employs xe2x80x9cnxe2x80x9d reaction vessels wherein nascent chains are extended by the addition respectively of the activated carbamates of xe2x80x9cnxe2x80x9d Boc-protected aza-amino acids. After each extension step, aliquots of the products are saved and cataloged and the remainder is pooled into a common pot to form a mixture. The mixture of nascent chains is then aliquoted into xe2x80x9cnxe2x80x9d reaction vessels for further extension. After xe2x80x9cmxe2x80x9d extensions, the product is decoupled and separated from the soluble support to form the combnatorial oligoazatide library.
Another aspect of the invention is directed to a combinatorial oligoazatide library. The library comprising a plurality of compounds represented by the following formula: 
wherein 0xe2x89xa6ixe2x89xa699 and Rx is selected from the group consisting of hydrogen, methyl, isobutyl, isopropyl, C1-C6 alkyl, benzyl, substituted benzyl and the side chain radical of the following amino acids: Ala, Arg, Asn, Asp, Asx, Cys, Gln, Glu, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.
Another aspect of the invention is directed to an azatide compounds represented by the following formulas: 
wherein R1, R2, R3 and R4 is selected from the group consisting of hydrogen, methyl, isopropyl, isobutyl and benzyl.
Another aspect of the invention is directed to intermediate oligoazatide compounds represented by the following formula: 
wherein 0xe2x89xa6ixe2x89xa699 and Rx is selected from the group consisting of hydrogen, methyl, isobutyl, isopropyl, C1-C6 alkyl, benzyl, substituted benzyl and the side chain radical of the following amino acids: Ala, Arg, Asn, Asp, Asx, Cys, Gln, Glu, Glx, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val.